Biology subjects

Imai, R.

Publications and source records attributed to Imai, R..

10 recordsLinked to original sources

bioRxiv2026

SAM-Targeted CRISPR-Cas9 RNP Delivery Combined with Leaf Regeneration Enables DNA-Free, Non-Chimeric Genome Editing in 'Fuji' Apple

DNA-free genome editing is a promising strategy for the genetic improvement of horticultural crops and fruit trees because it enables targeted mutagenesis without stable genetic transformation. In planta particle bombardment (iPB) delivers CRISPR-Cas9 ribonucleoproteins (RNPs) directly into shoot apical meristems (SAMs), enabling heritable genome editing without the use of tissue culture-based transformation systems. However, the practical application of iPB-mediated editing in fruit trees is limited by the frequent occurrence of chimerism, which cannot be readily eliminated through sexual segregation while maintaining the genetic background of elite cultivars. To overcome this limitation, we combined iPB-mediated RNP delivery with regeneration from edited leaf tissues (iPB-REG). Using this approach, we targeted the self-incompatibility gene S9-RNase in the elite apple cultivar Fuji and efficiently recovered non-chimeric edited plants. These results establish iPB-REG as a practical strategy for producing uniform genome-edited fruit trees and provide a valuable platform for DNA-free genetic improvement and functional genomics in clonally propagated perennial crops.

plant biology↗

bioRxiv2026

Evolutionary origin of prolonged delayed fertilization in the Fagaceae

The temporal organization of flowering, fertilization, and fruiting is a fundamental axis of life-history evolution in angiosperms. While most species complete fruit development within a single growing season ("1-year fruiting"), many Fagaceae species delay fertilization and fruit maturation until the year following flowering ("2-year fruiting"). Despite its ecological prevalence, the evolutionary origins of this strategy and its coordination with other functional traits remain poorly understood. In this study, we investigated the evolutionary origins of the two-year fruiting strategy by reconstructing ancestral states on a phylogeny comprising 88 species that represent all eight genera of Fagaceae. We further employed phylogenetic comparative analyses to test whether the fruiting trait is evolutionarily constrained by pollination mode (animal versus wind-pollinated) or leaf habit (evergreen versus deciduous). Our results support a single origin of 2-year fruiting in the common ancestor of the major clade excluding Fagus and Trigonobalanus, followed by multiple independent reversions to 1-year fruiting in lineages including Castanea, Quercus, and Castanopsis. Ancestral-state reconstructions of pollination mode and leaf habit strongly support entomophily and an evergreen leaf habit as ancestral traits of Quercoideae, predating the emergence of the 2-year fruiting strategy. Although entomophily and evergreen habit were ancestral in Quercoideae, our correlation analyses indicate that transitions in fruiting strategy did not depend on changes in these traits. These results provide macroevolutionary evidence that extreme extensions of the interval between pollination and fertilization can be evolutionarily stable yet rarely re-evolve once lost over long evolutionary timescales.

ecology↗

bioRxiv2025

Unconventional secretion of angiogenic sonic-hedgehog-containing large extracellular vesicles is stimulated via the PI3K/Rab18-GDP pathway

Large extracellular vesicles (lEVs), with diameters >600 nm, play essential roles in special types of intercellular communications. However, information on the molecular mechanism underlying their roles and on their relevance remains scarce. Here we show that PI3K-Rab18-GDP signaling facilitates the secretion of lEVs from human mesenchymal stem cells (hMSCs) and fibroblasts. These EVs contain a large amount of sonic hedgehog (SHH) protein and promote angiogenesis. Rab18 serves as the master facilitator of this secretion only in its GDP-bound form, which can be enriched by the Rab inhibitor CID1067700 or by the PI3K agonist SF1670. Rab18-GDP accumulates in the cell center and develops SHH-lEV precursors from perinuclear endosomes. Rab18-GDP recruits heat shock protein 90 (Hsp90) and neutral sphingomyelinase 2 (nSMase2) and facilitates vertical secretion of SHH-lEVs with an Hsp90 corona from the cell center. This secretory machinery may play an important role in developmental and regenerative PI3K-mediated angiogenesis.

cell biology↗

bioRxiv2024

A long shelf-life melon created via CRISPR/Cas9 RNP-based in planta genome editing

We developed a non-culture and DNA-free genome editing technique for melon using in plant Particle Bombardment (iPB-RNP). The method was employed to create CmACO1 mutants. One of the mutant lines exhibited an extended shelf life due to ethylene deficiency during fruit ripening. As no cell culture step is involved, the iPB-RNP method is expected to overcome limitations associated with conventional genome editing, such as genotype dependency and somatic variations. Thus, this methodology holds significant potential for application in commercial melon breeding and across a wide range of Cucurbitaceae species.

plant biology↗

bioRxiv2024

DNA-free and genotype-independent CRISPR/Cas9 system in soybean

The CRISPR/Cas9 system has revolutionized the field of plant genetic engineering. Here we report a smart genome editing system of soybean by using iPB-RNP method without introducing foreign DNA and requiring traditional tissue culture processes such as embryogenesis and organogenesis. Shoot apical meristem (SAM) of embryonic axes was used the target tissue for genome editing, because the SAM in soybean mature seeds has stem cells and specific cell layer developing germ cells during reproductive growth stage. In the iPB-RNP method, the complex of ribonucleoprotein (RNP) and Cas9 protein was directly delivered into SAM stem cells via particle bombardment and genome-edited plants were generated from these SAMs. Soybean allergenic gene Gly m Bd 30K, which we previously generated genome-editing soybean by using Agrobacterium-mediated transformation and particle bombardment in our previous studies, was targeted in this study. Many E0 (the first generation of genome-edited) plants in this experiment harbored mutant alleles at the targeted locus. Editing frequency of inducing mutations transmissible to the E1 generation was approximately 0.4 to 4.6 % of all E0 plants utilized in various soybean varieties. Furthermore, Gly m Bd 30K protein in mature seeds was not detected by western blot analysis due to flame-sift mutations. Our results offer a practical approach for both plant regeneration- and DNA-free genome editing achieved by delivering RNP into the SAM of dicotyledonous plants.

plant biology↗

bioRxiv2023

Intergenic regions contribute fitness advance by increasing recombination

Intergenic regions are common in eukaryotic genomes, yet they often appear to confer no direct fitness advantage. This study hypothesized that the primary function of these regions may be to enhance the frequency of meiotic recombination between genes, thereby reducing selective interference and improving the efficacy of selection. To test this hypothesis, computer simulations were conducted using the slim4 software to examine the impact of varying lengths of intergenic regions on total fitness. Populations with extended intergenic regions exhibited increased total fitness. Notably, the average fitness effects of both advantageous and deleterious mutations diminished, implying an enhanced selection efficiency, particularly for mutations with marginal fitness impacts. Total fitness effect of deleterious mutations increased despite the average fitness effect decreasing. Linkage between deleterious mutations could contribute to purging of slightly deleterious mutations.These results underscore the need for a more comprehensive examination of the relationship between recombination and intergenic regions to fully elucidate their evolutionary significance and contribution to genomic architecture. Article SummaryEvolutionary significance of intergenic regions are still unclear. This study hypothesized that the primary function of these regions may be to enhance the frequency of meiotic recombination between genes, thereby reducing selective interference and improving the efficacy of selection. The simulation showed that populations with extended intergenic regions exhibited increased total fitness. The results poin t the way for future research about evolution of intergenic regions and the effect of genomic position of genes.

evolutionary biology↗

bioRxiv2023

The molecular clock in long-lived tropical trees is independent of growth rate

The rates of appearance of new mutations play a central role in evolution. However, mutational processes in natural environments and their relationship with growth rates are largely unknown, particular in tropical ecosystems with high biodiversity. Here, we examined the somatic mutation landscapes of two tropical trees, Shorea laevis (slow-growing) and S. leprosula (fast-growing), in central Borneo, Indonesia. Using newly-constructed genomes, we identified a greater number of somatic mutations in tropical trees than in temperate trees. In both species, we observed a linear increase in the number of somatic mutations with physical distance between branches. However, we found that the rate of somatic mutation accumulation per meter of growth was 3.7-fold higher in S. laevis than in S. leprosula. This difference in the somatic mutation rate was scaled with the slower growth rate of S. laevis compared to S. leprosula, resulting in a constant somatic mutation rate per year between the two species. We also found that somatic mutations are neutral within an individual, but those mutations transmitted to the next generation are subject to purifying selection. These findings suggest that somatic mutations accumulate with absolute time and older trees have a greater contribution towards generating genetic variation. Significance StatementThe significance of our study lies in the discovery of an absolute time-dependent accumulation of somatic mutations in long-lived tropical trees, independent of growth rate. Through a comparative analysis of somatic mutation landscapes in slow- and fast-growing species, we observed a clock-like accumulation of somatic mutations in both species, regardless of their growth rates. Although the majority of somatic mutations were restricted to a single branch, we also identified mutations present in multiple branches, likely transmitted during growth. Our findings suggest that older trees make a greater contribution towards generating genetic variation.

evolutionary biology↗

bioRxiv2021

A ubiquitously expressed UDP-glucosyltransferase, UGT74J1, controls basal salicylic acid levels in rice

Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.

plant biology↗

bioRxiv2021

In planta genome editing with CRISPR/Cas9 ribonucleoproteins

In planta genome editing represents an attractive approach to engineering crops/varieties that are recalcitrant to culture-based transformation methods. Here, we report the direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem using in planta particle bombardment and introduction of a semidwarf1 (sd1)-orthologous mutation into wheat. The triple knockout tasd1 mutant of an elite wheat variety reduced culm length by 10% without a reduction in yield.

plant biology↗

bioRxiv2019

A novel defective recombinant porcine enterovirus G virus carrying a porcine torovirus papain-like cysteine protease gene and a putative anti-apoptosis gene in place of viral structural protein genes

Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3 kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9 kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G.

microbiology↗